Method for screening ameliorants of dry skin caused by atopic dermatitis using bleomycin hydrolase activity as indicator

ABSTRACT

The present invention provides a method for screening and evaluating ameliorants of dry skin caused by atopic dermatitis, comprising: evaluating a candidate drug as being an ameliorant of dry skin caused by atopic dermatitis in the case the candidate drug significantly increases expression and/or activity of bleomycin hydrolase in comparison with a control drug.

TECHNICAL FIELD

The present invention provides a method for screening and evaluatingameliorants of dry skin, and particularly dry skin caused by atopicdermatitis, a method for improving or preventing dry skin caused byatopic dermatitis, and a method for diagnosing dry skin caused by atopicdermatitis.

BACKGROUND ART

Keratin fibers present in the granular layer of the epidermis aggregateby binding to a protein referred to as filaggrin during keratinizationand produce a characteristic morphology referred to as a “keratinpattern”. Although a precursor substance of filaggrin known asprofilaggrin (consisting of an arrangement of 10 to 12 filaggrin units)is present in large amounts in keratohyalin granules within granularcells, together with the formation of filaggrin monomers, keratin fibersare caused to aggregate by dephosphorylation during keratinization.Subsequently, the aggregated keratin fibers are subjected to deiminationby the action of an enzyme known as peptidyl arginine deiminase (PAD),are released as keratin, and are subsequently decomposed to amino acidsand the like in the upper layer of the horny layer. These amino acidsare referred to as natural moisturizing factors (NMF), play an importantrole in maintaining the moisture content of the horny layer, and areknown to possess the ability to absorb ultraviolet light (Blank, I. H.,J.I. Dermatol., 18, 433 (1952); Blank, I. H., J.I. Dermatol., 21, 259(1953)).

Ever since amino acids functioning as the main component of NMF weredetermined to originate in filaggrin, research has been conducted on thecorrelation between disease states presenting with dry skin andfilaggrin. Amino acid levels in the horny layer have recently beendetermined to decrease in dry skin associated with conditions such assenile xerosis or atopic diseases (Horii, I. et al., Br. J. Dermatol.,121, 587-592 (1989); Tanaka, M. et al., Br. J. Dermatol., 139, 618-621(1989)).

PAD deiminates filaggrin by acting on arginine residues, and convertsthem to citrulline residues. As a result of filaggrin being deiminatedin this manner, the affinity between filaggrin and keratin fibersweakens and the keratin fibers are released, and as a result thereof,filaggrin becomes susceptible to the action of proteases, and this isultimately thought to lead to its decomposition to NMF.

The inventor of the present invention identified calpain 1 as an enzymethat decomposes filaggrin deiminated by PAD, and determined that thedecomposition products thereof in the form of small peptide fragmentsare decomposed to amino acid units, namely NMF, by bleomycin hydrolase(BH) (Journal of Investigative Dermatology (2008), Volume 128,Abstracts, 590, 539; Joint Conference of the 30th Annual Meeting of theMolecular Biology Society of Japan and 80th Annual Scientific Meeting ofthe Japanese Biochemical Society, Collection of Abstracts, p. 533;Journal of Biological Chemistry, 284, No. 19, pp. 12829-12836, 2009,3P-0251; and, Japanese Patent Application No. 2008-135944 (to bereferred to as JP944).

More recently, some atopic dermatitis is known to be caused by a geneticabnormality of the profilaggrin gene, and this genetic abnormality isobserved in roughly 5% to 50% of atopic dermatitis patients (Smith, F.J. D., et al., Nat. Genet. 38: 337-342 (2006): Aileen Sandilands, atal., J.I. Dermatol., 127, 1282-1284 (2007); and, Nomura, T. et al., J.I.Dermatol., 128(6): 1436-41 (2008)). However, the skin of atopicdermatitis patients is not necessarily associated with a dramaticdecrease in expression of filaggrin.

PRIOR ART DOCUMENTS Non-Patent Documents

Non-Patent Document 1: Blank, I. H., J.I. Dermatol., 18, 433 (1952)

Non-Patent Document 2: Blank, I. H., J.I. Dermatol., 21, 259 (1953)

Non-Patent Document 3: Horii, I. et al., Br. J. Dermatol., 121, 587-592(1989)

Non-Patent Document 4: Tanaka, M. et al., Br. J. Dermatol., 139, 618-621(1989)

Non-Patent Document 5: Kamata, et al., J. Biochem., 141, 69-76 (2007)

Non-Patent Document 6: Journal of Investigative Dermatology (2008),Volume 128, Abstracts, S90, 539

Non-Patent Document 7: Joint Conference of the 30th Annual Meeting ofthe Molecular Biology Society of Japan and 80th Annual ScientificMeeting of the Japanese Biochemical Society, Collection of Abstracts, p.583, 3P-0251

Non-Patent Document 8: Journal of Biological Chemistry, 284; No. 19, pp.12829-12836, 2009

Non-Patent Document 9: Smith, F. J. D. et al., Nat. Genet. 38: 337-42(2006)

Non-Patent Document 10: Aileen Sandilands, et al., J.I. Dermatol., 127,1282-1284

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method for screeningdrugs that improve or prevent dry skin caused by atopic dermatitis basedon a novel mechanism of the occurrence of rough skin attributable tofluctuations in expression of an NMF-producing enzyme, a method forevaluating dry skin caused by atopic dermatitis, a method for improvingor preventing dry skin caused by atopic dermatitis, and a method fordiagnosing dry skin caused by atopic dermatitis.

Means for Solving the Problems

In the aforementioned JP944, the inventors of the present inventiondetermined that increased activity of bleomycin hydrolase improves thebarrier function of skin through the production of NMF. In this manner,bleomycin hydrolase is thought to be act in the final stage of NMFproduction. However, it is interesting to note with respect to dry skinoccurring due to atopic dermatitis that, since expression of filaggrincontinues to be observed in numerous atopic dermatitis patients, thisphenomenon is predicted to be caused by that other than an abnormalityof the filaggrin gene.

Based on the hypothesis that decreased expression of bleomycin hydrolasein human skin is not only related to a decrease in the skin's barrierfunction due to abnormality in the mechanism by which NMF is produced,but also to atopic dermatitis primarily caused by an immune disorder ordry skin and the like caused by atopic dermatitis, the inventors of thepresent invention verified fluctuations in the expression of this enzymeby conducting tests on dry skin in humans and analyzed the mechanism bywhich its expression is controlled. As a result, the inventors of thepresent invention found that decreased expression of bleomycin hydrolaseis related to dry skin caused by atopic dermatitis, and that a controlregion that clearly induces expression of the enzyme is present in the5′ flanking region of a gene that encodes that enzyme. Morespecifically, the inventors of the present invention cloned the 5′flanking region of BH. A region important for BH promoter activity wasidentified −216 bp upstream in a deletion analysis thereof. Anelectrophoretic mobility shift assay clearly demonstrated that MZF-1,Sp-1 and interferon regulatory factor (IRF)-1/2 are able to bind to thisregion in vitro. Moreover, BH promoter activity decreased considerablywhen a site-specific mutation was induced in the MZF-1 and Sp-1 motifs.These data suggested that BH expression is up-regulated through MZF-1and Sp-1. It is interesting to note that that the Th1 cytokine,interferon (IFN)-γ significantly decreased expression of BH. Theinhibitory effect of IFN-γ on BH expression was demonstrated in ananalysis using site-specific mutagenesis and small interfering RNA.Although the Th2 cytokine, IL-4, did not demonstrate any direct actionwhatsoever on BH expression, it down-regulated MZF-1 and Sp-1 incultured keratinocytes. Thus, this suggested that IL-4 is able to act asa suppressor of BH regulation. Finally, the expression of BH wasinvestigated in the skin of patients suffering from AD. Since BHactivity and expression decreased considerably in skin affected by AD, adefect was suggested to be present in the filaggrin decompositionpathway in AD. As has been described above, the inventors of the presentinvention found that transcription of BH is likely regulated both duringdifferentiation and inflammation, thereby leading to completion of thepresent invention.

Thus, the present application includes the inventions indicated below.

-   (1) A method for screening and evaluating ameliorants of dry skin    caused by atopic dermatitis, comprising evaluating a candidate drug    as being an ameliorant of dry skin caused by atopic dermatitis in    the case the candidate drug significantly increases expression    and/or activity of bleomycin hydrolase in comparison with a control    drug.-   (2) The method of (1), wherein expression and/or activity of    bleomycin hydrolase is judged to have increased significantly in the    case the transcription activity of a gene that encodes bleomycin    hydrolase has increased significantly in comparison with that of a    control.-   (3) The method of (2), wherein the transcription activity is judged    to have increased significantly in the case binding activity of    transcription factors IRF-1, IRF-2, MZF-1, Sp-1 and/or GATA-1 to the    transcription regulatory region of a gene that encodes bleomycin    hydrolase has increased significantly in comparison with that of a    control.-   (4) A method for improving or preventing dry skin caused by atopic    dermatitis by significantly increasing expression and/or activity of    bleomycin hydrolase in skin tissue.-   (5) The method of (4), wherein the expression and/or activity is    significantly increased by increasing the transcription activity of    a gene that encodes bleomycin hydrolase.-   (6) The method of (5), wherein the expression and/or activity is    significantly increased by increasing binding activity of    transcription factors IRF-1, IRF-2, MZF-1, Sp-1 and/or GATA-1 to the    transcription regulatory region of a gene that encodes bleomycin    hydrolase.-   (7) A method for diagnosing predisposition to dry skin caused by    atopic dermatitis, comprising diagnosing a tendency towards dry skin    caused by atopic dermatitis in the case expression and/or activity    of bleomycin hydrolase in skin tissue is significantly decreased in    comparison with that of control skin, while diagnosing the absence    of a tendency towards dry skin caused by atopic dermatitis if it is    equal to or greater than that of the control skin.-   (3) The method of (7), wherein the expression and/or activity is    judged to be significantly decreased in the case the transcription    activity of a gene that encodes bleomycin hydrolase is significantly    decreased in comparison with that of a control.-   (9) The method of (8), wherein the transcription activity is judged    to be decreased in the case binding activity of transcription    factors IRF-1, IRF-2, MZF-1, Sp-1 and/or GATA-1 is significantly    decreased in comparison with that of a control.

Effects of the Invention

Novel ameliorants of dry skin caused by atopic dermatitis can be foundbased on the novel mechanism of the occurrence of dry skin establishedby the present invention attributable to fluctuations in the expressionof bleomycin hydrolase, and particularly on a screening system that usesexpression and/or activity of bleomycin hydrolase as an indicator.Moreover, the method of the invention of the present application is alsoconsidered to be applicable to searching for ameliorants of ordinary dryskin not caused by atopic dermatitis. In fact, as described in examplesof the present application, in a group demonstrating low levels ofexpression and/or activity of bleomycin hydrolase, the barrier functionof the skin (transepidermal water loss: TEWL) decreased significantlyalong with a decrease in horny layer moisture content.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Western blots indicating the relationship between theamount of bleomycin hydrolase and the number of tape strippings in humanskin extracts obtained by tape stripping.

FIG. 2 shows a western blot indicating the relationship between theamount of bleomycin hydrolase and dry skin in human skin extracts. T andA represent samples obtained from subjects not sensing dryness, Nrepresents a sample obtained from a subject sensing some degree ofdryness, and M represents a sample obtained from a subject sensingdryness.

FIG. 3 is a graph indicating the relationship between the amount ofbleomycin hydrolase and the enzyme activity thereof in horny layerextracts obtained from human arms. The numbers on the horizontal axisindicate subject identification numbers.

FIG. 4 indicates a first order approximation of the relationship betweenthe amounts and activities of bleomycin hydrolase obtained in FIG. 3 asdetermined according to the least-squares method.

FIG. 5 indicates statistical analyses relating to bleomycin hydrolasepresent in horny layer extracts obtained from human arms and skinparameters (A: free amino acids, B: activity, C: TEWL). BH low:bleomycin hydrolase level<10, activity<1.5 (nmol/min/ml); BH high:bleomycin hydrolase level≧10, activity≧1.5 (nmol/min/ml).

FIG. 6 is a flow chart of a survey used to classify skin.

FIG. 7 shows the results of measuring skin parameters of the horny layerobtained from subjects classified according to the flow chart of FIG. 6.

FIG. 8 shows tissue staining images indicating the localization ofbleomycin hydrolase and filaggrin in normal skin.

FIG. 9 shows tissue staining images indicating the localization ofbleomycin hydrolase and filaggrin in the skin of a patient with atopicdermatitis.

FIG. 10 is a graph indicating the relationship between thedifferentiation of keratinocytes and expression level of bleomycinhydrolase as determined using quantitative PCR. Values on the verticalaxis represent relative amounts based on a value of 1 for the expressionlevel at 80% confluence.

FIG. 11 is a schematic diagram showing a 5′ flanking region of a genethat encodes bleomycin hydrolase.

FIG. 12 is a graph indicating the results of a luciferase assay of BHpromoters using human epidermal keratinocytes.

FIG. 13 shows graphs indicating the relationship between expression oftranscription factors SP1, MZF-1 and GATA-1 and UV irradiation.

FIG. 14 shows graphs indicating the relationship between the expressionlevels of bleomycin hydrolase and proteases and UV irradiation in normalhuman epidermal keratinocytes.

FIG. 15 shows primers used to prepare continuous 5′-deletion mutants ofa 5′ flanking region of BH by PCR.

FIG. 16 shows primers used to analyze transcription levels of BH andrelated factors by quantitative real-time RT-PCR.

FIG. 17 shows probes used to analyze electrophoretic mobility shift.

FIG. 18A shows a schematic diagram of a 5′ flanking region of human BH.Putative transcription factor binding sites within the 5′ flankingregion were determined by a search using the Genome Net MOTIF program.FIG. 18B shows BH promoter regions as determined by deletion analysis.In FIG. 18C, putative transcription factor binding sites indicating thenucleotide sequence of a region from −216 to −1 that includes theminimum BH promoter sequence and the putative transcription bindingsites.

FIG. 19A indicates characterization of transcription factor bindingsites in BH promoters by site-specific mutagenesis, a schematic diagramof deletion constructs of putative transcription factor binding sites,and the luciferase activity thereof in cultured keratinocytes.Site-specific mutagenesis was carried out in constructs spanning thenucleotide sequence of the region from −616 to +1. FIG. 19B indicatesbinding of MZF-1, Sp-1, GATA-1 or IRF-1/2 to the cis-acting element ofBH promoters. The experiment was carried out with an electrophoreticmobility shift assay (EMSA) using a nuclear extract from culturedkeratinocytes and a biotinated double-stranded oligonucleotide probecontaining putative transcription factor binding site MZF-1, Sp-1,GATA-1 or IRF-1/2. Lane 1 indicates the binding profile of thebiotinated probe in the nuclear extract, while lane 2 indicates thebinding profile of the biotinated probe after competing with anon-labeled probe present in an excess amount equal to twice the amountof the biotinated probe.

FIG. 20A shows the results of real-time RT-PCR analyses of BHexpression. The graphs indicate the effects of cytokines Th1, Th2 andTh17 on BH gene expression. FIG. 20B shows the results of mutationanalyses of IRF-1/2 binding sites. The graphs indicate BH promoteractivity in cultured keratinocytes in the presence of IFN-γ.Keratinocytes were transfected with pGL3-216 containing intact IRF-1/2binding sites of BH promoter regions followed by treating for 24 hourswith IFN-γ (upper panel). Keratinocytes were transfected with ΔpGL3-616(IRF-1/2 deletion mutants) followed by treating for 24 hours in thepresence or absence of IFN-γ or IL-4 at 10 ng/ml (lower panel). FIG.20(C) shows the results of measuring IRF-1 and IRF-2 gene expressionlevels using small interfering RNA (siRNA) to judge whether or notIRF-1/2 are essential mediators of IFN-γ-induced down-regulation of BH.Keratinocytes were transfected with siRNA (40 nM) of IRF-1 or IRF-2followed by culturing for 24 hours, treating with IFN-γ at 10 ng/ml andisolating RNA after further culturing for 24 hours. The panel on theright indicates the silencing effects of IRF-1 and IRF-2.

FIG. 21A shows the results of an expression analysis of BH, calpain andputative transcription factors in proliferative or differentiated cellsby real-time PCR to investigate the mechanism of transcription controlin the epidermis. FIG. 21B shows the results of an analysis of theexpression patterns of transcription factors MZF-1, Sp-1, GATA-1, IRF-1and IRF-2 in cultured keratinocytes.

FIG. 22A indicates the effects of IFN-γ on expression of putativetranscription factors IRF-1 and IRF-2. FIG. 22B indicates the effects ofIL-4 on expression of putative transcription factors IRF-1, IRF-2, MZF-1and Sp-1.

FIG. 23A indicates the simultaneous localization of BH and filaggrin inthe granular layer as demonstrated by double staining with anti-BHantibody and anti-filaggrin antibody in normal epidermis. FIG. 23Bindicates the BH activities of extracts obtained from affected skin andunaffected skin of an AD patient.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect thereof, the present invention provides a method forscreening and evaluating ameliorants of dry skin caused by atopicdermatitis, comprising evaluating a candidate drug as being anameliorant of dry skin caused by atopic dermatitis in the case thecandidate drug significantly increases expression and/or activity ofbleomycin hydrolase in comparison with a control drug.

Bleomycin hydrolase is a cytoplasmic cysteine peptidase having amolecular weight of 250 kDa to 280 kDa (hexamer), and was initiallyknown to have the function of metabolic deactivation of the glycopeptidebleomycin frequently used in cancer combination chemotherapy. Bleomycinhydrolase contains an active site residue characteristic of the papainsuperfamily of cysteine proteases, and its encoding gene is present atgene locus 17q11.2 in humans (Takeda, et al., J. Biochem., 119, 29-36,1996). It is present in all tissues, and although it is also known to bepresent in skin (Kamata, et al., J. Biochem., 141, 69-76, 2007), itsrelationship with filaggrin was completely unknown prior to thedetermination thereof by the inventor of the present invention.

Based on the results of tissue staining, bleomycin hydrolase wasdetermined to be expressed in large amounts in the upper layer of theepidermis in normal skin in the same manner as filaggrin (FIG. 8). Onthe other hand, expression of bleomycin hydrolase and filaggrindecreases at the sites of atopic rashes in atopic dermatitis patients(FIG. 9). This strongly suggests that an abnormality in this enzymesystem, and not an abnormality of the profilaggrin gene, is the cause ofatopic dermatitis. In addition, bleomycin hydrolase activity issignificantly lower in not only affected areas, but also unaffectedareas, in the skin of atopic dermatitis patients (data not shown).

Moreover, as a result of studying fluctuations in expression levels ofbleomycin hydrolase using cultured keratinocytes, in contrast to thisenzyme not being expressed at a high level in undifferentiatedkeratinocytes, it was found to be highly expressed in differentiatedkeratinocytes that had reached confluence, hardly expressed at all inbasal cells, and highly expressed in cells in which differentiation hadprogressed and had transformed into epidermal cells (FIG. 10). Thisresult supports the result obtained from cell staining as previouslydescribed.

Measurement of the expression and/or activity of bleomycin hydrolaseaccording to the present invention can be carried out quantitatively orqualitatively in accordance with any arbitrary method able to be used tomeasured the expression and/or activity of this enzyme, such as animmunoassay method that utilizes antibody specific to bleomycinhydrolase, examples of which include ELISA using an enzyme label, RIAusing a radioactive label, immunonephelometry, western blotting, latexagglutination and hemagglutination. Examples of types of immunoassaysinclude competitive assays and sandwich assays. More specifically,measurement of the aforementioned activity can be carried out by, forexample, utilizing the property of citrulline of being a substratenearly specific to bleomycin hydrolase, and evaluating the decompositionof its fluorogenic substrate, Cit-MCA, with a fluorescencespectrophotometer. Measurement of the amount of bleomycin hydrolase canalso be carried out by measuring the expression level of a gene thatencodes the enzyme. In this case, the expression level of bleomycinhydrolase is preferably determined by measuring the amount of mRNA thatencodes bleomycin hydrolase within cells. Extraction of mRNA andquantitative or qualitative measurement of the amount thereof are knownin the art, and can be carried out by various known methods, such asPCR, 3SR, NASBA or TMA. In addition, bleomycin hydrolase can also bequalitatively determined by in situ hybridization or through measurementof the biological activity thereof.

In the method for screening and evaluating ameliorants of dry skincaused by atopic dermatitis of the present invention, a candidate drugis evaluated as being an ameliorant of dry skin caused by atopicdermatitis in the case the candidate drug significantly increases theexpression and/or activity of bleomycin hydrolase in comparison with acontrol drug. Drugs screened in accordance with the method of thepresent invention are considered to be effective for dry skin, andparticularly for dry skin caused by atopic dermatitis.

According to guidelines for the examination and treatment of atopicdermatitis of the Japan Dermatological Association, “atopic dermatitis”is defined as a disease associated with the primary complaint of itchyeczema that undergoes repeated exacerbation and improvement, and can bediagnosed based on the presence or absence of itching, and in terms ofthe characteristic rash thereof, the presence of acute lesions such aserythema, weeping erythema, papules, serous papules, scales or scabs, aswell as chronic lesions such as infiltrative erythema, lichenifiedlesions, prurigo, scales or scabs. When used in the present description,“dry skin caused by atopic dermatitis” refers to dry skin associatedwith atopic dermatitis that has been definitively diagnosed according tothe aforementioned definition, including genetic abnormalities of thefilaggrin gene.

Examples of the screening method of the present invention include amethod consisting of measuring an increase in the expression of mRNAthat encodes bleomycin hydrolase in the presence of a drug to be tested,and a method consisting of inserting a promoter sequence involved inexpression of bleomycin hydrolase into a luciferase gene vector, anddirectly measuring the degree of expression in the form of a promoterassay system. In the case of the latter, a region from −216 bp to −816bp at which BH expression reaches a maximum is preferably used for theBH promoter region. In addition, a fluorescent protein such asAzami-Green commonly used in the art can be used to measure expressionlevel instead of luciferase. The action of a drug on BH expression canbe measured by introducing a vector containing a fused gene thereof intocells, culturing the cells in the presence of a drug, lysing the cellsnormally after 24 hours and measuring luciferase activity. Althoughcommercially available normal human epidermal keratinocytes (NHEK, suchas those available from Kurabo Industries, Ltd.) or immortalized HaCaTcells and the like can be used for the cells used for measurement, thecells used are not limited thereto. Measurement of luciferase activityis preferably carried out by using a luciferase assay kit such as thatavailable from Roche Diagnostics K.K.

When used, in the present description, “significantly increasesexpression and/or activity of bleomycin hydrolase in comparison with acontrol drug” refers to the case in which the measured expression level,activity or both of bleomycin hydrolase is 120% or more, 150% or more,or 200% or more, respectively, in comparison with a drug that does nothave an ameliorative effect on dry skin, and, particularly anameliorative effect on dry skin caused, by atopic dermatitis.

Increasing the expression and/or activity of bleomycin hydrolase canalso be achieved by increasing the transcription activity of a gene thatencodes the enzyme. A 5′ flanking region of a gene that encodesbleomycin hydrolase, and particularly a transcription regulatory region,and transcription factors that bind to that region, are shown in FIG.11. The aforementioned transcription regulatory region has a regionextending 216 bp to 1216 bp downstream from the sequence that encodesthe enzyme. The transcription regulatory region preferably contains aregion extending 816 bp downstream from the viewpoint of obtaining ahigh level of bleomycin hydrolase activity.

A region extending at least 216 bp downstream from the sequence encodingbleomycin hydrolase is to be contained in order to express this enzyme.Among the transcription factors described in FIG. 11, expression ofbleomycin hydrolase is thought to be particularly enhanced by increasingthe binding activities of IRF-1, IRF-2, MZF-1, Sp-1 and GATA-1 containedin this region. In fact, when expression of bleomycin hydrolase isincreased by irradiating with ultraviolet (UV) light (data not shown), acorrelation is observed between expression levels of MZF-1 and GATA-1and UV intensity and irradiation time (FIG. 13).

Here, in the case the transcription activity of a gene encodingbleomycin hydrolase, or the binding activity of a transcription factorto the transcription regulatory region of that gene, is significantlyincreased by a candidate drug, such as by 120% or more, 150% or more or200% or more, respectively, in comparison with a control drug, theexpression level and/or activity of bleomycin hydrolase can beconsidered to be significantly increased.

Expression and/or activity of bleomycin hydrolase are also affected bycytokines. For example, interleukin-4 (IL-4), which is known to beinvolved in atopic dermatitis and is a type of Th2 cytokine,down-regulates expression of bleomycin hydrolase. This coincides withthe low expression level of bleomycin hydrolase observed in the skin ofatopic dermatitis patients. On the other hand, interferon-γ, which is atypical Th1 cytokine that has the opposite action of IL-4 of inhibitingproduction of IgE, significantly increases expression of bleomycinhydrolase. In addition, tumor necrosis factor-alpha (TNF-α), which is aTh2 cytokine representative of inflammatory cytokines, alsosignificantly increases expression of this enzyme. In addition to thesesubstances, expression and/or activity of bleomycin hydrolase is alsoincreased by UV irradiation. Although the results thereof are not shown,the activity of bleomycin hydrolase in the skin of the cheeks or otherlocation on the body susceptible to ultraviolet irradiation has beenconfirmed to be increased by UV irradiation.

In a second aspect thereof, the present invention provides a method forimproving or preventing dry skin caused by atopic dermatitis bysignificantly increasing expression and/or activity of bleomycinhydrolase in skin tissue.

In the method for improving or preventing dry skin caused by atopicdermatitis of the present invention, the expression and/or activity ofbleomycin hydrolase in the skin is significantly increased in comparisonwith, for example, the expression and/or activity in skin prior toundergoing this treatment method. “Significantly increased” refers tothe case in which, for example, the expression and/or activity ofbleomycin hydrolase is made to be a value of 120% or more, 150% or moreor 200% or more.

In the method of the present invention, an arbitrary drug is used thatsignificantly increases the expression and/or activity of bleomycinhydrolase. In addition, there are no limitations on the drug provided itincreases the expression and/or activity of this enzyme. The drug andthe like used in the method of the present invention can be applied tothe skin in an arbitrary form provided it can be applied to skin and itis able to achieve the object of the present invention, and the drug maybe applied alone or may be applied by incorporating with other arbitrarycomponents. In addition, there are no limitations an the location of theskin where the drug is applied, and includes any skin on the bodysurface, including the scalp.

In a third aspect thereof, the present invention provides a method fordiagnosing predisposition to dry skin caused by atopic dermatitis,comprising diagnosing a tendency towards dry skin caused by atopicdermatitis in the case expression and/or activity of bleomycin hydrolasein skin tissue is significantly decreased in comparison with that ofcontrol skin, while diagnosing the absence of a tendency towards dryskin caused by atopic dermatitis if it is equal to or greater than thatof the control skin.

Whether or not the skin of a subject is dry can be judged based on thesubjectivity of the subject or physician and the like, or can be judgedobjectively by measuring the moisture content of skin using a skinsurface moisture analyzer. For example, as explained in Experiment 3 ofthe present description, whether or not a subject has dry skin can alsobe judged according to the subjectivity of a subject based on oiliness,susceptibility to drying and the like in accordance with the flow chartdescribed in FIG. 6.

Although dry skin can be easily assessed, judging whether the skin of asubject is predisposed to dry skin caused by atopic dermatitis may bedifficult. According to the diagnostic method of the present invention,a diagnosis can be made not only of the current status of a subject'sskin, but also as to whether or not the subject is susceptible to dryskin caused by atopic dermatitis.

“Expression and/or activity of bleomycin hydrolase in skin tissue issignificantly decreased in comparison with that of control skin” refersto the case in which measured expression and/or activity of bleomycinhydrolase is 80% or less, 70% or less, 60% or less, 50% or less, 30% orless or 10% or less in comparison with, for example, normal “controlskin” judged to be moist skin by a physician from a dermatologicalperspective. “Equal to or greater than that of the control skin” refersto the case in which measured expression and/or activity of bleomycinhydrolase is, for example, 80% or more, 90% or more or 100% or more incomparison with, for example, normal “control skin” judged to be moistskin by a physician from a dermatological perspective.

Although collection of a skin horny layer sample to serve as a specimencan be carried out by an arbitrary method, tape stripping is preferablefrom the viewpoint of convenience. Tape stripping refers to a method bywhich a horny layer sample is collected by affixing a piece ofpressure-sensitive adhesive tape to the skin surface layer and peelingoff the tape so that the skin horny layer adheres to the peeledpressure-sensitive adhesive tape. Use of the tape stripping method makesit possible to measure expression and activity of bleomycin hydrolasesimply by sampling the horny layer with a single piece of tape, therebyenabling non-invasive evaluation of dry skin caused by atopic dermatitisusing bleomycin hydrolase as an indicator. In a preferable methodemploying tape stripping, the surface layer of the skin is first cleanedwith ethanol, for example, to remove sebaceous matter, dirt and thelike, a piece of pressure-sensitive adhesive tape cut to a suitable size(such as 5 cm×5 cm) is gently placed on the skin surface, the entirepiece of tape is pressed flat onto the skin surface by applying uniformpressure, and the pressure-sensitive adhesive tape is subsequentlypeeled off while applying uniform force. The pressure-sensitive adhesivetape may be commercially available cellophane tape, and examples ofpressure-sensitive adhesive tape that can be used include Scotch SuperStrength Mailing Tape(3M) and Cellophane Tape (CelloTape®, Nichiban).

The following provides a more detailed explanation of the presentinvention by listing specific examples thereof. Furthermore, the presentinvention is not limited by these specific examples.

EXAMPLES

The following materials were used in the experiments.

Calpain I was purchased from EMD Biosciences Inc. Bleomycin hydrolasewas prepared from human epidermal horny layer in accordance withNon-Patent Document 5. Human IL-4 and IFN-γ were purchased fromPeprotech EC (London, England). Human IL-13 and IL-17A/F were producedby R&D Systems Inc. (Minneapolis, Minn.).Citrulline-4-methylcoumaryl-7-amide (Cit-MCA) was acquired from BachemBioscience Inc. (Bubendorf, Switzerland). Reagent grade products wereused for all other chemical substances used.

Culturing of Keratinocytes

Normal human epidermal keratinocytes derived from neonatal epidermis(Kurabo, Osaka, Japan) were cultured in EpiLife medium (CascadeBiologics, Portland, Oreg.) containing low-concentration (0.03 mM)calcium and HKGS Growth Supplement (Cascade Biologics). All cells wereincubated at 37° C. while supplying 5% CO₂ and were used within 4passages. Cells were collected at 70% confluence, 100% confluence, 2days after confluence and 2 days after confluence in 2 mM calcium.

Experiment 1

Bleomycin hydrolase is thought to act at the final stage of NMFproduction. In this case, there is the possibility of the expression ofthis enzyme decreasing in dry skin. In this experiment, a study was madeas to whether or not a decrease in the expression and/or activity ofbleomycin hydrolase in skin is related to dry skin.

Epidermal horny layer samples were collected by tape strippingconsisting of affixing transparent pressure-sensitive adhesive tape(CelloTape®, Nichiban) to a skin surface on the arm followed by peelingoff the tape. The tape adhered with the epidermal horny layer was cutinto pieces, immersed in an extraction buffer (0.1 M Tris-HCl (pH 8.0),0.14 M NaCl, 0.1% Tween-20, 1 ml), and subjected to ultrasonic treatmentto prepare a horny layer extract. The horny layer extract was thensubjected to western blotting. The anti-bleomycin hydrolase (BH)antibody used was prepared in accordance with the method of Kamata, etal. (Journal of Biological Chemistry, 2009). More specifically, thehorny layer extract was subjected to SDS electrophoresis followed bytransferring to Immobilon-P (Millipore), and after washing thetransferred film, was allowed to react for 1 hour at room temperaturewith the anti-BH antibody. After removing the antibody by additionalwashing, the film was allowed to react with HRP-bound secondaryantibody. After washing, the BH protein bands that were made to beluminescent with the ECL Plus Western Blotting Detection Kit (GEHealthcare) were printed onto an X-ray film, and expression levels wereestimated based on the intensity thereof. The results are shown in FIGS.1 and 2.

In FIG. 1, specimen 1 is a skin horny layer sample of a subject who wasthought to have dry skin at the subject's own discretion, while specimen2 is a skin horny layer sample of a healthy student not thought to havedry skin. In addition, specimens T and A in FIG. 2 are from subjects whodid not perceive to have dry skin, specimen N was from a subject whoperceived to have somewhat dry skin, and specimen M was from a subjectwho perceived to have dry skin. The expression level of bleomycinhydrolase in specimen 1 is low, while the expression level thereof inspecimen 2 is high. On the basis thereof, specimens 1 and 2 can bedetermined to have been derived from dry skin and moist skin,respectively. In addition, based on the results using specimen 1, in dryskin the amount of bleomycin hydrolase can be determined to decreasemore the closer to the skin surface serving as the production site ofNMF. In the western blot of FIG. 2, specimens T and A indicate extractsobtained from subjects that did not particularly perceive dry skin,while specimens N and M are extracts from subjects who were stronglyaware of having dry skin.

Experiment 2

In this experiment, a study was made of individual differences in theamount and activity of bleomycin hydrolase in human skin and thecorrelation between the amount and activity of this enzyme. Horny layerextracts were prepared from the skin of 40 female students ranging inage from 20 to 25 years old in accordance with the method described inExperiment 1. The amounts of bleomycin hydrolase in the extracts and theactivity thereof were measured in accordance with the method of Kamata,at al. (J. Biol. Chem., Vol. 284, Issue 19, 12829-12836, May 8, 2009).Expression levels were evaluated by western blotting, whileaminopeptidase activity of the enzyme was evaluated by measuring thedecomposed amount of Cit-β-NA used for the fluorogenic substrate. Theresults are shown in FIG. 3, and a correlation diagram thereof is shownin FIG. 4. As is clear from the results depicted in FIG. 4, acorrelation exists between the amount of bleomycin hydrolase and theactivity thereof.

Then, a statistical analysis was carried out on bleomycin hydrolase andvarious skin parameters for the aforementioned horny layer extracts. Inthis experiment, the horny layer extracts from 40 subjects wereclassified into the following two types. After converting the amounts ofbleomycin hydrolase determined from the results of western blotting intonumerical values with a densitometer, extracts having an amount ofbleomycin hydrolase of less than 10 in the case of using a value of “1”as an arbitrary unit and having enzyme activity of less than 1.5nmol/min/ml were classified as having a low protein level of bleomycinhydrolase and low activity (BH low), while all other extracts wereclassified as having a high protein level and high activity (BH high).

Free amino acids were measured in accordance with the method of Kamata,et al. (J. Biol. Chem., Vol. 284, Issue 19, 12829-12836, May 8, 2009).More specifically, filaggrin peptide decomposed with calpain I wasallowed to react with each extract followed by quantifying the amount ofamino groups using Fluorescamine and measuring the amount of free aminoacids. The results of measuring free amino acids are shown in FIG. 5A.The units of the vertical axis in FIG. 5A represent the total amount offree amino acids (nmol) in 3 ml of measurement sample.

As was previously described, bleomycin hydrolase activity was evaluatedas the aminopeptidase activity of the enzyme by measuring the decomposedamount of Cit-β-NA serving as a fluorogenic substrate. The results ofmeasuring bleomycin hydrolase activity are shown in FIG. 5B. The unitsof the vertical axis in FIG. 5B represent the decomposed amount ofCit-β-NA (nmol/min/ml).

TEWL levels in the skin of the aforementioned students were measuringusing a Vapometer (Delfin Technologies, Ltd., Finland), and wereindicated in g/m²/h. The results of measuring TEWL levels are shown inFIG. 5C.

As shown in FIG. 5C, a significant difference in horny layer moisturelevels was present between the group having low bleomycin hydrolaseactivity (2.5 U<) and the high group. Moreover, in the group in whichboth the amount and activity of this enzyme were low, free amino acidlevels were low and TEWL levels were high (FIGS. 5A and 5C).

Although the data is not shown, significant differences in the amountsof NMF and urocanic acid were present between the low free amino acidgroup (1000<) and the high free amino acid group, while a significantdifference in urocanic acid levels was present between the low NMF group(0.8<) and the high NMF group. In addition, significant differences inNMF, lactic acid and urea were present between the low TEWL group (2.5<)and the high TEWL group. When considering that urocanic acid is producedfrom histidine that is present in large amounts in filaggrin, bleomycinhydrolase can be determined to play an important role in thedecomposition of filaggrin.

On the basis of the results of this experiment, both free amino acidlevels and barrier function can be determined to decrease significantlyin the case of a low absolute amount of bleomycin hydrolase. Althoughthe data is not shown, even in the case of using a horny layer extractderived from the cheek, a proportional relationship was confirmed toexist between the amount of bleomycin hydrolase and barrier function.

Experiment 3

In this experiment, a survey was conducted among the aforementionedfemale students based on the flow chart shown in FIG. 6, and the skin ofthe students was classified into one of four categories consisting ofmoist skin, dry skin, dry oily skip and oily skin. The results of thissurvey and the correlation with the results for skin parameters measuredin the aforementioned Experiment 2 are shown in FIG. 7. On the basis ofFIG. 7, bleomycin hydrolase activity of students classified as havingoily dry skin was determined to be significantly high.

Experiment 4

In this experiment, a study was made of the localization of bleomycinhydrolase and filaggrin in the skin.

Immunohistochemical Staining

Immunohistochemical staining was carried out according to the method ofKamata, at al. (J. Biol. Chem., Vol. 284, Issue 19, 12829-12836, May 8,2009). Frozen sections of human skin having a thickness of 5 μm andanti-rat BH IgG were used for the samples. More specifically, human skinspecimens were obtained from patients suffering from atopic dermatitisat the Tokyo Medical University after obtaining their informed consent.This study was approved by the Tokyo Medical University InstitutionalReview Board and a Shiseido Special Subcommittee with respect to humanethics.

Sections of human atopic dermatitis (affected skin and unaffected skin)and normal skin were incubated for 1 hour at room temperature withanti-rat BH IgG and anti-human filaggrin IgG, followed by washing withPBS and further incubating with a fluorescent bound secondary antibodyin the form of Alexa Fluor 555 or 488 (Molecular Probes Inc., Eugene,Oreg.). DAPI (4′,6′-diamidino-2-phenylindole, Molecular Probes) was usedto visualize the nucleus.

The results of tissue staining normal skin are shown in FIG. 8, whilethe results of comparing skin from a healthy subject (normal skin) andskin from a patient with atopic dermatitis (location of atopic rash) areshown in FIG. 9. As shown in FIG. 9, bleomycin hydrolase was shown to behighly expressed in the upper layer of the epidermis and localized inthe same manner as filaggrin. On the other hand, at locations of atopicrash, expression of bleomycin hydrolase and filaggrin were low incomparison with normal skin (FIG. 9).

Quantitative PCR

The expression level of bleomycin hydrolase in keratinocytes wasmeasured by quantitative PCR using the method described below.Measurement was carried out using the Light Cycler 480 (RocheDiagnostics GmbH, Mannheim, Germany), while Light Cycler FastStart DNAMaster CYBR Green I was used for the reagent. 0.6 μl aliquots of each ofthe following bleomycin hydrolase primers and 6.9 μl of water were addedto 10 μl of SYBR Green I master mix to bring to a total volume of 20 μlfollowed by carrying out 45 cycles of PCR consisting of 15 seconds at95° C., 20 seconds at 55° C. and 20 seconds at 72° C. The resultsobtained were corrected by comparing with the results for a housekeepinggene in the form of G3PDH.

Forward primer: (SEQ ID NO. 1) TGTGGTTTGGCTGTGATGTT Reverse primer:(SEQ ID NO. 2) GCACCATCCTGATCATCCTT

The results of the aforementioned quantitative PCR are shown in FIG. 10.As shown in FIG. 10, bleomycin hydrolase was expressed higher inkeratinocytes that had reached confluence, namely differentiatedkeratinocytes, in comparison with keratinocytes at 80% confluence,namely undifferentiated keratinocytes. In other words, on the basis ofthe results of this experiment, this enzyme was determined to not beexpressed at high levels in basal cells prior to differentiation. Theresults of this quantitative PCR support the results obtained in theaforementioned tissue staining.

Experiment 5

1) Lucerifase Assay of BH Promoters Using Human Epidermal Keratinocytes

Lysis buffer (200 μl) was added to proliferative phase keratinocytes(approx. 80% confluence) or differentiated keratinocytes (after reachingconfluence, exposing to air and adding 2 mM calcium followed bycontinuing to culture for 2 days) to lyse the cells. The Bright-GloLuciferase Assay System (Promega Co., Madison, Wis., USA) was used formeasurement. 20 μl of sample were transferred to a prescribed tubefollowed by measurement using the Auto Lumat Plus (L89538, Berthold GmbH& Co., KG, Bad Wildbad, Germany). Based on the results shown in FIG. 12,it was determined that in order to express bleomycin hydrolase, theaforementioned transcription regulatory region must have a regionextending at least 216 bp downstream from the sequence encoding theenzyme.

2) UV Irradiation of NHEK

RNA was collected by a prescribed method at 3 hours, 24 hours and 48hours after irradiating with UVB at 30 mJ or 60 mJ (TorexF120S-E-30/DMR, 20 W, Toshiba Medical Supply), and mRNA expressionlevels of bleomycin hydrolase and calpain were measured by quantitativePCR. As a result, the sample collected 48 hours after irradiating at 30mJ expressed the highest level of bleomycin hydrolase (FIG. 13).

3) Effect of Cytokines on Bleomycin Expression

IL-4 (final concentration: 0.1, 1.0 or 10 ng/ml), TNF-α (finalconcentration: 0.1, 1.0 or 10 ng/ml) and IFN-γ (final concentration:1.0, 10 or 100 ng/ml) were respectively added to proliferative stagecultured keratinocytes, and after incubating for 24 hours, RNA wascollected using Isogen. Expression of bleomycin hydrolase mRNA wasmeasured by quantitative PCR. The results'are shown in FIG. 14. Based onthe results shown in FIG. 14, interleukin-4 (IL-4), which is a type ofTh2 cytokine, was determined to down-regulate expression of bleomycinhydrolase.

Experiment 6

Characterization of Human BH Gene

1) Cloning of BH 5′ Flanking Region

The 5′ flanking region of BH was amplified based on the nucleotidesequence of human BH gene using the Genome Walker Kit (Clontech,Mountain View, Calif.) in accordance with the manufacturer's protocol byusing a gene-specific primer 1 (GSP1) having the sequence5′-tcctcgagtctgtatcagagcagctaca-3′ (SEQ ID NO. 3) and a gene-specificprimer 2 (GSP) having the sequence 5′-tgaacacgcgtccgagctgctcatggcg-3′(SEQ ID NO. 4). In brief, primary PCR was carried out using Ex Taq DNAPolymerase (Takara, Shiga, Japan) in the presence of 5%dimethylsulfoxide by using GSP1 and an adapter primer (AP) 1 and using atwo-step PCR protocol recommended by the manufacture consisting of 7cycles of 25 seconds at 94° C. and 4 minutes at 72° C. followed by 32cycles of 25 seconds at 94° C. and 4 minutes at 67° C., and finallyelongation for 4 minutes at 67° C. Next, the primary PCR mixture wasdiluted and used as a template of secondary PCR amplification using GSP2and AP2. Secondary PCR was carried out in the same manner as primary PCRwith the exception of using 5 cycles instead of 7 cycles for the initialnumber of cycles and using 20 cycles instead of 32 cycles for thesubsequent number of cycles. Continuous 5′-deletion mutants of the 5′flanking region of BH were produced by PCR using the primers shown inFIG. 15. Following amplification, all of the PCR products were cloned inpGEM-T Easy Vector (Promega, Madison, Wis.) followed by sequencedetermination using the ABI Prism 310 Genetic Analyzer (AppliedBiosystems, Foster City, Calif.).

In order to construct a reporter plasmid pGL3-1216/+1, PCR was carriedout under the conditions of 30 cycles of initial denaturation for 4minutes at 94° C., 30 seconds at 94° C. and 1 minute at 72° C. followedby final elongation for 4 minutes at 72° C. using as templatespGEM-T-1216/+1 along with a pair of specific BH primers(5′-ccgggtaccatcagagttccttagaa-3′ (SEQ ID NO. 5) and5′-taaatacgcgttggcgcccacgctgccg-3′ (SEQ ID NO. 6)) containingrestriction sites KpnI and MluI. The resulting PCR mixture was digestedwith KpnI and MluI and cloned in pGL3-Basic vector (Promega).Furthermore, the pGL3-Basic vector contains firefly luciferase gene. Allof the constructs were prepared using the Qiagen Plasmid Midi Kit(Qiagen, Dusseldorf, Germany).

2) Site-Specific Mutagenesis

Mutagenesis of MZF-1, Sp-1 and IRF-1/2 binding sites was carried out byusing the Quick Change Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.) in accordance with the manufacturer's protocol. Primersconsisting of 5′-ggaccccgtttcagcctccccgcc-3′ (SEQ ID NO. 7) (forwardprimer of mutant Sp-1 site) and 5′-ggcggggaggctgaaacggggtcc-3′ (SEQ IDNO. 8) (reverse primer of mutant Sp-1 site) were used to produce adeletion mutation in Sp-1. Primers consisting of5′-gactcagcaacgcggttttgtccctccgc-3′ (SEQ ID NO, 9) (forward primer ofmutant MZF-1 site) and 5′-gcggagggacaaaaccgcgttgctgagtca-3′ (SEQ ID NO.10) (reverse primer of mutant MZF-1 site) were used for the MZF-1mutant. Primers consisting of 5′-gccgccgagcctccggcgctcc-3′ (SEQ ID NO.11) (forward primer of mutant IRF-1/2 site) and5′-ggagcgccggaggctcggcggc-3′ (SEQ ID NO. 12) (reverse primer of mutantIRF-1/2 site) were used for the IRF-1/2 mutant.

3) Transfection and Measurement of Promoter Activity

Keratinocytes were cultured in a 12-well tissue culture plate at adensity 5×10⁴ cells/well, and were transfected using FuGene HDTransfection reagent (Roche Diagnostics, Basel, Switzerland) and 1 μgaliquots of each construct. All of the cells were simultaneouslytransfected with pGL4.74 [hRluc-TK] vector (Promega) containing seapansy (Renilla) luciferase under the control of an HSV-TK promoter forthe purpose of correcting transfection efficiency. Unless specificallyindicated otherwise, the cells were collected 24 hours aftertransfection and lysed using the Passive Lysis buffer (Promega) at 250μl per well. Luciferase activity was analyzed using the Dual LuciferaseReporter Assay System (Promega) and Autolumat Plus Luminometer (BertholdTechnologies, Bad Wildbad, Germany). Firefly luciferase activity wasstandardized based on the sea pansy luciferase activity. Threetransfection procedures were carried out independently on eachconstruct, and the results were represented in the form of an averagevalue.

4) Quantitative Real-Time RT-PCR Analysis

The transcription levels of BH and related factors were analyzed byquantitative real-time RT-PCR. Total RNA was extracted from culturedcells using Isogen (Nippon Gene, Tokyo, Japan) in accordance with themanufacturer's protocol. The total RNA was reverse-transcribed to cDNAusing SuperScript™ II (Invitrogen, Carlsbad, Calif.). Real-time RT-PCRwas carried out with the LightCycler Raid Cycler System using theLightCycler 480 SYBR Green I Master (Roche Diagnostics) in accordancewith the manufacturer's protocol. Information relating to the primersused is shown in FIG. 16. Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was used as a reference gene. Specificity of the amplifiedfragments was confirmed by quantitative analysis of melting curves usingLightCycler analytical software. The amounts of mRNA were standardizedbased on the mRNA of GAPDH and finally indicated as a ratio to mRNA ofan untreated control.

5) siRNA-Based Inhibition of IRF-1 and IRF-2

Cultured keratinocytes were transfected using Lipofectamine RNAi Max(Invitrogen, Carlsbad, Calif.) with 40 nM siIRF-1, siIRF-2 and siControlA (Santa Cruz Biotechnology, Santa Cruz, Calif.) in accordance with themanufacturer's protocol. The cells were then cultured for 24 hours inantibody-free media, followed by extracting the total RNA and analyzingby real-time RT-PCR in the manner previously described.

6) Electrophoretic Mobility Shift Analysis (EMSA)

Double-stranded oligonucleotide probes were prepared by annealingsingle-stranded biotinated oligonucleotides with single-strandedunlabeled oligonucleotides (FIG. 17). Nuclear extraction and EMSA werecarried out using a Nuclear Extraction Kit and EMSA Gel Shift Kit(Panomics, Santa Clara, Calif.). The nuclear extracts (4 μg) wereincubated for 30 minutes at 15° C. with 1× binding buffer, 1 μg ofPoly[d(IC)] and biotinated probes (50 pmol) corresponding to the MZF-1,Sp-1, IRF-1/2 and GATA-1 binding sites. In order to carry out acompetitive assay, a two-fold excess amount of unlabeled probe was addedto binding reaction prior to addition of the biotinated probes. Theseincubation mixtures were then electrophoresed with 0.5× TBE buffer in 8%polyacrylamide gel followed by transfer to a Biodyne B Nylon membrane(Pall, Port Washington, N.Y.). The bands were visualized by using thechemiluminescence detection kit provided in the EMSA Gel Shift Kit.

7) Results

Isolation and Characterization of Human BH Gene Promoters

A large number of putative transcription factor binding sites have beendetermined to be present within the 5′ flanking region of human BH basedon the results of a search using the Genome Net MOTIF program (FIG.16A). In particular, since sequences showing close coincidence withconsensus sequences recognized by MZF-1, Sp-1, IRF-1/2 and GATA-1/2 arepresent in a region extending from −216 to +1 near the locations oftranscription initiation sites, these transcription factors weresuggested to be involved in the regulation of BH promoter activity. Moreprecisely, a deletion analysis was carried out to determine BH promoterregions (FIG. 18B). The highest level of luciferase activity wasdetected in differentiated keratinocytes transfected with pGL3-816.However, the relative luciferase activity of the deletion plasmidsremained high until deletion proceeded to pGL3-216. Among theconstructs, the plasmid containing the fragment extending from −444 to+1 (indicated as pGL3-444) demonstrated significantly lower activity inthe cultured keratinocytes, and upstream suppressor activity wassuggested to be present in the −616 to −444 region. Since these resultsdemonstrated that the region from −216 to −1 contains the minimalpromoter for BH gene transcription, the nucleotide sequence thereof isshown in FIG. 18C. Since this sequence did not contain a TATA or CCAATbox, it was suggested to possess housekeeping properties of this gene.On the other hand, several transcription factor binding sites, such asMZF-1, Sp-1, IRF-1/2 and GATA-1/2, were present in this core promoterregion.

Identification of Latent Cis-Acting Element Involved in

BH Gene Regulation

A new series of deletion mutants was constructed targeted at eachcis-acting element to determine the latent cis-acting element of theminimal promoter involved in regulating transcription of BH geneexpression. Promoter activity was greatly down-regulated in the case ofdeleting the MZF-1, Sp-1 and IRF-1/2 binding sites (FIG. 19A).

Moreover, en investigation was conducted as to whether or not thesetranscription factors are actually able to bind to each of the putativebinding sites. Therefore, an electrophoretic mobility shift assay (EMSA)was carried out using a nuclear extract from cultured keratinocytesalong with double-stranded oligonucleotide probes containing the MZF-1,Sp-1, GATA-1 or IRF-1/2 binding site. As shown in FIG. 19B, althoughSp-1, MZF-1 and IRF-1/2 bound to the target site corresponding to BHpromoter, GATA-1/2 did not bind. These results indicate that thesebinding sites of the promoter region extending from −216 to −105 bp areessential for the cis-acting element for BH transcription.

Cytokine-Mediated Regulation of BH Gene Expression

Since BH is an NMF-generating enzyme, it has the potential to beinvolved in the pathophysiology of AD. Accordingly, an investigation wasmade of the effects of cytokines Th1, Th2 and Th17 on EH geneexpression. FIG. 20A indicates that the Th1 cytokine, IFN-γ,down-regulated expression of BH mRNA in a dose-dependent manner inproliferative keratinocytes. On the other hand, cytokines Th2 and Th17did not demonstrate any significant effects whatsoever on BH expression.Similar results were obtained with differentiated keratinocytes (datanot shown). A promoter assay was carried out in order to determine therole of IFN-γ in regulation of BH gene expression, and response elementswere specified. As shown in FIG. 20B, IFN-γ down-regulated BH promoteractivity in proliferative keratinocytes transfected with pGL3-BH-616containing an IRF-1/2 binding sequence between −131 and −120. Followingdeletion of this sequence, IFN-γ no longer inhibited BH promoteractivity (FIG. 20B). In addition, IRF-1 and IRF-2 gene expression wasinhibited using small interfering RNA (siRNA) in order to determinewhether or not IRF-1/2 is an essential mediator of IFN-γ-induceddown-regulation of BH. IFN-γ activity was significantly inhibited incultured keratinocytes transfected with either IRF-1 or IRF-2 siRNA (40nM) (FIG. 20C). These results strongly suggest that the IRF-1/2 bindingsequence is essential for IFN-γ-induced down-regulation of BH geneexpression.

Expression of BH and Related Factors in Cultured Keratinocytes

The expression of BH, calpain-1 and putative expression factors inproliferative and differentiated cells was analyzed by real-time PCR inorder to investigate the mechanism of transcription regulation in theepidermis. As shown in FIG. 21A, BH mRNA was up-regulated indifferentiated keratinocytes, such as in those 2 days after theconfluent stage (3.6 times) and those cultured at a high calciumconcentration (8.6 times), in comparison with proliferativekeratinocytes. These results coincide with the promoter assay data (FIG.18B). Similar results were also obtained with respect to calpain I(roughly 2.5-fold increase in up-regulation). In addition, aninvestigation was made of expression vectors of various transcriptionfactors such as MZF-1, Sp-1, GATA-1, IRF-1 and IRF-2 in proliferativekeratinocytes. As shown in FIG. 21B, these transcription factors wereup-regulated in differentiated keratinocytes in line with BH expression.However, expression of GATA-1 mRNA was significantly lower in comparisonwith other factors (<1/32). GATA-1 is thought not to play an importantrole in keratinocytes. Accordingly, BH is suggested to be synthesized ina differentiation-dependent manner mediated by MZF-1 and Sp-1. The factto that IRF-1 and IRF-2 are also up-regulated by differentiation stimuliindicates that BH expression is extremely sensitive to IFN-γ.

Effects of Cytokines Th1 and Th2 on Expression of Putative TranscriptionFactors

An investigation was made of the cytokine-dependent regulation of thesetranscription factors. FIG. 22A indicates that IFN-γ stronglyup-regulates expression of IRF-1 mRNA in a dose-dependent manner.Similarly, expression of IRF-2 was up-regulated in the presence ofIFN-γ. In contrast, expression of IRF-1 and IRF-2 was significantlyenhanced only in the presence of IL-4 at 100 ng/ml (FIG. 22B). It isinteresting to note that both MZF-1 and Sp-1 were most effectivelydown-regulated in the presence of IL-4 at 10 ng/ml (FIG. 22C). Theseresults suggest that expression of BH is regulated directly andindirectly by cytokines Th1 and Th2, respectively.

Down-Regulation of BH in Atopic Dermatitis Skin

Although loss-of-function mutations of FLG are related to the mechanismof occurrence of AD, there is the possibility of this being related notto a gene deletion, but rather to a disorder in the decompositionpathway or the pathology of AD. Consequently, an investigation was nextmade of the localization of BH and filaggrin in affected skin andunaffected skin of AD patients along with BH activity at thoselocations. In normal skin, double staining with anti-BH antibody andanti-filaggrin antibody demonstrated simultaneous localization of BH andfilaggrin in the upper epidermis, and particularly in the granularlayer, as previously reported (FIG. 23A). At a higher magnification,although BH was clearly indicated to be localized from the granularlayer to the horny layer, filaggrin was limited to granular cells. Incontrast, BH expression decreased dramatically in the affected skin andunaffected skin of AD patients observed in this study (n=7). All ofthese results indicated that, even though significant staining wasdetected at all times, comparatively weak filaggrin staining wasindicated (FIG. 23A). In addition to immunohistochemistry, BH activitywas measured in horny cell extracts from tape peeling samples obtainedfrom 18 AD patients and 30 healthy volunteers. The extracts obtainedfrom affected skin and unaffected skin of the AD patients demonstratedsubstantially lower BH activity than that of the healthy volunteers(27.1% and 8.8% lower, respectively) (FIG. 28B). These resultsdemonstrated that BH is simultaneously localized with filaggrin, andthat the activity thereof is dramatically decreased in the skin ofpatients suffering from AD.

Discussion

In this study, the regulatory mechanism of BH gene expression wasexamined by cloning and functional characterization of the promoterregion. A region important for BH promoter activity was identified to bepresent in a region located −216 bp upstream by promoter analysis (FIG.18B). In this region, putative MZF-1 and Sp-1 binding sites demonstrateda significant effect on BH promoter activity (FIGS. 18C and 19A). It isinteresting to note that Sp-1 and MZF-1 have also been reported to beinvolved in the regulation of PAD1 that is an important enzyme forinitiation of filaggrin decomposition. Sp-1 is a typical member of theSp/Kruppel-like family of zinc finger proteins that function astranscription factors in mammalian cells. It is thought to be involvedin nearly all aspects of cell function, including proliferation,apoptosis, differentiation and neoplastic transformation. In humanepidermis, Sp-1 is an important regulatory factor of genes thatparticipate in epidermal differentiation, including the involucrin,loricrin, transglutaminase, and PAD1, PAD2 and PAD3 genes. MZF-1 is atranscription factor that belongs to the Krupple family of zinc fingerproteins, and is expressed in differentiated totipotent hematopoieticcells and bone marrow progenitor cells. However, the function of MZF-1during transcription regulation in mammalian epidermis has not beenreported. MZF-1, Sp-1 and BH were found to be simultaneouslyup-regulated in differentiated keratinocytes in comparison withproliferative keratinocytes (FIG. 21B), indicating the role of BH indifferentiation rather than housekeeping. These results clearlyindicated that these transcription factors function as activatingfactors for basic regulation of transcription of BH during finaldifferentiation of keratinocytes.

On the other hand, an investigation of cis-acting elements furtherdefined the IRF-1/2 binding sites within this region. The IRFs wereconfirmed to bind directly to the BH promoter region by using EMSA (FIG.19B). Site-specific mutagenesis of this binding region brought about asignificant decrease in BH promoter activity (FIG. 13A). Consequently,IRF-1/2 transcription factors are also most likely required for minimalpromoter activity of the BH gene under basic conditions. The IRF familyconsists of a group of transcription factors, and at present, nine IRFmembers (IRF-1 through IRF-9) have been identified in various cell typesand tissues. These IRF molecules play a role in antiviral defense,immune response/regulation and cell growth regulation when stimulated byIFN-α, IFN-β and IFN-γ. IRF-1 and IRF-2 have been demonstrated tofunction as agonists and antagonists involved in the regulation ofnumerous IFN-γ-induced genes. It is interesting to note that IFN-γremarkably decreased expression of BH mRNA (FIGS. 20A and 20B).Knockdown and site-specific mutagenic analyses confirmed that IRF-1/2binding sites are involved in IFN-γ-mediated inhibition of BH expression(FIGS. 20B and 20C). These results clearly indicate that IRF-1/2 aremediators of IFN-γ-mediated down-regulation of BH gene in humankeratinocytes. On the other hand, the Th2 cytokines of IL-4 and IL-13did not demonstrate any direct action whatsoever during incubation for24 hours (FIG. 20A). However, these Th2 cytokines significantlyinhibited expression of activator molecules in the form of MZF-1 andSp-1. Accordingly, it is reasonable to think that Th2 cytokinesdown-regulate expression of BH.

In addition, BH was shown to be dramatically down-regulated in affectedand unaffected AD skin (FIGS. 23A and 23B). Although filaggrin mutationis a primary risk factor for diseases related to barrier disorders suchas AD, mutation analyses indicated that this mutation accounts for lessthan 50% of the incidences of these diseases in Ireland and no more than20% of the incidences in Japan. Defective synthesis of filaggrin as wellas disorders associated with filaggrin decomposition are hypothesized tobe involved in a breakdown of barrier function. It is clear thatdecreased levels of NMF bring about dry skin, and that this promotes abreakdown of the skin barrier. AD is well known to be a Th2-polarizeddisease. However, recent reports have suggested that Th1 cytokines alsoplay a role in AD. For example, “intrinsic AD” is immunologicallycharacterized by low expression levels of IL-4, IL-5 and IL-13 and ahigh expression level of IFN-γ. In addition, a shift from Th1 to Th2occurs during a transition from the acute phase to the chronic phase ofAD skin. These results also indicate the possibility that IFN-γ may playa more important role than was previously thought.

In conclusion, these results indicate that BH transcription in humanepidermis is regulated by two modes of regulation. The first pathway isunder the control of final keratinocyte differentiation, while the otherpathway is dependent on cytokines Th1 and Th2. Since these pathways areinterrelated, the balance there between is likely to easily shifttowards down-regulation of BH expression. Since a decrease in BH bringsabout a shortage of NMF, this leads to dry skin and eventually asubsequent breakdown of barrier function in response thereto. Theseresults provide novel findings regarding the regulation of BH and themechanism of occurrence of AD.

1. A method for screening and evaluating ameliorants of dry skin causedby atopic dermatitis, comprising: evaluating a candidate drug as beingan ameliorant of dry skin caused by atopic dermatitis in the case thecandidate drug significantly increases expression and/or activity ofbleomycin hydrolase in comparison with a control drug.
 2. The methodaccording to claim 1, wherein expression and/or activity of bleomycinhydrolase is judged to have increased significantly in the case thetranscription activity of a gene that encodes bleomycin hydrolase hasincreased significantly in comparison with that of a control.
 3. Themethod according to claim 2, wherein the transcription activity isjudged to have increased significantly in the case binding activity oftranscription factors IRF-1, IRF-2, MZF-1, Sp-1 and/or GATA-1 to thetranscription regulatory region of a gene that encodes bleomycinhydrolase has increased significantly in comparison with that of acontrol.
 4. A method for improving or preventing dry skin caused byatopic dermatitis by significantly increasing expression and/or activityof bleomycin hydrolase in skin tissue.
 5. The method according to claim4, wherein the expression and/or activity is significantly increased byincreasing the transcription activity of a gene that encodes bleomycinhydrolase.
 6. The method according to claim 5, wherein the expressionand/or activity is significantly increased by increasing bindingactivity of transcription factors IRF-1, IRF-2, MZF-1, Sp-1 and/orGATA-1 to the transcription regulatory region of a gene that encodesbleomycin hydrolase.
 7. A method for diagnosing predisposition to dryskin caused by atopic dermatitis, comprising: diagnosing a tendencytowards dry skin caused by atopic dermatitis in the case expressionand/or activity of bleomycin hydrolase in skin tissue is significantlydecreased in comparison with that of control skin, while diagnosing theabsence of a tendency towards dry skin caused by atopic dermatitis if itis equal to or greater than that of the control skin.
 8. The methodaccording to claim 7, wherein the expression and/or activity is judgedto be significantly decreased in the case the transcription activity ofa gene that encodes bleomycin hydrolase is significantly decreased incomparison with that of a control.
 9. The method according to claim 8,wherein the transcription activity is judged to be decreased in the casebinding activity of transcription factors IRF-1, IRF-2, MZF-1, Sp-1and/or GATA-1 is significantly decreased in comparison with that of acontrol.